Polymer drug delivery systems have been developed for the controlled release of pharmaceutical polypeptides. For example, synthetic polyesters such as poly(DL-lactic acid), poly(glycolic acid), poly(lactic-glycolic acid), and poly(xcex5-caprolactone) have been used in the form of microcapsules, films, or rods to release biologically active polypeptides. See e.g., U.S. Pat. Nos. 4,767,628 and 4,675,189 and PCT Application No. WO 94/00148.
In addition to the synthetic polymeric chains, natural polymers and their derivatives have been used as components in similar sustained release compositions that dissociate by enzymatic degradation. One example of such natural polymers are those based on chitin, a poly(N-acetylglucosamine). However, since chitin is water insoluble, others have examined solubilizable derivatives which are based primarily on a partially deacetylated chitin, e.g., chitosan. See e.g., Sanford, P. A. et al., Eds., Advances in Chitin and Chitosan (1992). Although chitosan can be found in some fungi, the production of biodegradable chitosan is generally performed synthetically. See Mima, et. al., J. Appl. Polym. Sci. 28:1909-1917 (1983). Synthetic derivatives of chitosan have also been prepared to alter the polymer""s in vivo biological characteristics. See Muzzarelli, et al., Carbohydrate Res. 207:199-214 (1980).
The use of chitin, as well as chitin derivatives, has been proposed in a number of drug delivery systems. See. e.g., European Patent Application Nos. 486,959, 482,649, 525,813 A1, and 544,000 A1; and U.S. Pat. No. 5,271,945.
In one aspect, the present invention features a copolymer including an N-acylated derivative of poly(2-amino-2-deoxy-D-glucose), wherein between 1 and 50 percent of the free amines of the poly(2-amino-2-deoxy-D-glucose) are acylated with a first acyl group, the first acyl group is COE1 where E1 is selected from the group consisting of C3-33 carboxyalkyl, C3-33 carboxyalkenyl, C7-39 carboxyarylalkyl, and C9-39 carboxyarylalkenyl, and between 50 and 99 percent of the free amines of the poly(2-amino-2-deoxy-D-glucose) are acylated with a second acyl group, the second acyl group is COE2 where E2 is selected from the group consisting of C1-30 alkyl, C2-30 alkenyl, C6-37 arylalkyl, and C8-37 arylalkenyl, provided at least one of the free amines of the derivative is acylated with the first acyl group.
The copolymer preferably has a molecular weight of about 3,000 to 90,000 daltons. In other preferred embodiments, over 90 percent of the free amines of the poly(2-amino-2-deoxy-D-glucose) are acylated with either the first acyl group or the second acyl group. Preferably, between 10 and 30 percent of the free amine of the poly(2-amino-2-deoxy-D-glucose) are acylated with the first acyl group. Some of the free hydroxy groups (e.g., between 1 and 30 percent) of the derivative may be acylated with either the first acyl group or the second acyl group.
In a preferred embodiment, the copolymer is of the formula: 
wherein:
R1, for each individual repeat unit, is selected from the group consisting of first acyl group, second acyl group, and H;
R2, for each individual repeat unit, is selected from the group consisting of first acyl group, second acyl group, and H;
R3, for each individual repeat unit, is selected from the group consisting of first acyl group, second acyl group, and H;
R4 is selected from the group consisting of first acyl group, second acyl group, and H;
R5 is selected from the group consisting of first acyl group, second acyl group, and H;
R6 is selected from the group consisting of first acyl group, second acyl group, and H;
R7 is selected from the group consisting of COH and CH2OR8;
R8 is selected from the group consisting of first acyl group, second acyl group, and H;
n is between 2 and 200; and
for between 1 and 50 percent of the repeat units, R1, is first acyl group, and for between 50 and 99 percent of the repeat units, R1 is second acyl group, provided that for at least one of the repeat units, R1 is first acyl group.
The terms COE1 and COE2 stand for xe2x88x92Cxe2x95x90O.E1 and xe2x88x92Cxe2x95x90O.E2, respectively. The substituents carboxyalkyl, carboxyalkenyl, carboxyarylalkyl, and carboxyarylalkenyl may contain 1-4 carboxylic acid functionalities. Examples of the first acyl group include, but are not limited to, succinyl, 2-(C1-30 alkyl)succinyl, 2-(C2-30 alkenyl)succinyl, maleyl, phthalyl, glutaryl, and itaconyl. Examples of the second acyl group include but are not limited to, acetyl, benzoyl, propionyl, and phenylacetyl.
The present invention also features a composition including the above copolymer and a polypeptide, the polypeptide comprising at least one effective ionogenic amine, wherein at least 50 percent, by weight, of the polypeptide present in the composition is ionically bound to the polymer. Preferably, the composition comprises between 5 and 50 percent, by weight, of the polypeptide.
Preferred embodiments of the present invention include a copolymer wherein the first acyl group is succinyl and the second acyl group is acetyl and R7 is COH or CH2OH; a composition comprising said copolymer of claim 1 and H-xcex2-D-Nal-Cys-Tyr-D-Trp-Lys-Val-Cys-Thr-NH2 or a pharmaceutically acceptable salt thereof, wherein the two Cys are bonded by a disulfide bond, where at least 50 percent, by weight, of H-xcex2-D-Nal-Cys-Tyr-D-Trp-Lys-Val-Cys-Thr-NH2 or a pharmaceutically acceptable salt thereof, present in said composition is ionically bound to said copolymer; a composition comprising the foregoing copolymer and a peptide selected from the group consisting of 
or a pharmaceutically acceptable salt thereof, where at least 50 percent, by weight, of said peptide or a pharmaceutically acceptable salt thereof present in said composition is ionically bound to said copolymer; a composition comprising the foregoing copolymer and a peptide selected from the group consisting of (p-Glu-His-Trp-Ser-Tyr-D-Trp-Leu-Arg-Pro-Gly-NH2), ([D-Ser(t-Bu)6, des-Gly-NH210]-LHRH(1-9)NHEt), ([D-Trp6, des-Gly-NH210]-LHRH(1-9)NHEt, ([des-Gly-NH210]-LHRH(1-9)NHEt), ([D-Ser(t-Bu)6, Azgly10]-LHRH), ([D-His(Bzl)6, des-Gly-NH210]-LHRH(1-9)NHEt), ([D-Leu6, des-Gly-NH210]-LHRH(1-9)NHEt), ([D-Trp6, MeLeu7, des-Gly-NH210]-LHRH(1-9)NHEt), and ([D-Nal6]-LHRH, or a pharmaceutically acceptable salt thereof, where at least 50 percent, by weight, of said peptide or a pharmaceutically acceptable salt thereof, present in said composition is ionically bound to said copolymer; a composition comprising the foregoing copolymer and parathyroid hormone, an analogue thereof or a pharmaceutically acceptable salt thereof, where at least 50 percent, by weight, of parathyroid hormone, an analogue thereof or a pharmaceutically acceptable salt thereof, present in said composition is ionically bound to said copolymer.
Further preferred embodiments of the present invention include a copolymer wherein the first acyl group is glutaryl and the second acyl group is propionyl and R7 is COH or CH2OH; a composition comprising the foregoing copolymer and H-xcex2-D-Nal-Cys-Tyr-D-Trp-Lys-Val-Cys-Thr-NH2, wherein the two Cys are bonded by a disulfide bond, where at least 50 percent, by weight, of H-xcex2-D-Nal-Cys-Tyr-D-Trp-Lys-Val-Cys-Thr-NH2, present in said composition is ionically bound to said copolymer; a composition comprising the foregoing copolymer and a peptide selected from the group consisting of 
or a pharmaceutically acceptable salt thereof, where at least 50 percent, by weight, of said peptide or a pharmaceutically acceptable salt thereof present in said composition is ionically bound to said copolymer; a composition comprising the foregoing copolymer and a peptide selected from the group consisting of (p-Glu-His-Trp-Ser-Tyr-D-Trp-Leu-Arg-Pro-Gly-NH2), ([D-Ser(t-Bu)6, des-Gly-NH210]-LHRH(1-9)NHEt), ([D-Trp6, des-Gly-NH210]-LHRH(1-9)NHEt, ([des-Gly-NH210]-LHRH(1-9)NHEt), ([(D-Ser(t-Bu)6, Azgly10]-LHRH), ([D-His(Bzl)6, des-Gly-NH210]-LHRH(1-9)NHEt), ([D-Leu6, des-Gly-NH210]-LHRH(1-9)NHEt), ([D-Trp6, MeLeu7, des-Gly-NH210]-LHRH(1-9)NHEt), and ([D-Nal6]LHRH, or a pharmaceutically acceptable salt thereof, where at least 50 percent, by weight, of said peptide or a pharmaceutically acceptable salt thereof, present in said composition is ionically bound to said copolymer; and a composition comprising the foregoing copolymer and parathyroid hormone, an analogue thereof or a pharmaceutically acceptable salt thereof, where at least 50 percent, by weight, of parathyroid hormone, an analogue or a pharmaceutically acceptable salt thereof, present in said composition is ionically bound to said copolymer.
Examples of suitable polypeptides include growth hormone releasing peptide (GHRP), luteinizing hormone-releasing hormone (LHRH), somatostatin, bombesin, gastrin releasing peptide (GRP), calcitonin, bradykinin, galanin, melanocyte stimulating hormone (MSH), growth hormone releasing factor (GRF), growth hormone (GH), amylin, tachykinins, secretin, parathyroid hormone (PTH), encephalon, endothelin, calcitonin gene releasing peptide (CGRP), neuromedins, parathyroid hormone related protein (PTHrP), glucagon, neurotensin, adrenocorticothrophic hormone (ACTH), peptide YY (PYY), glucagon releasing peptide (GLP), vasoactive intestinal peptide (VIP), pituitary adenylate cyclase activating peptide (PACAP), motilin, substance P, neuropeptide Y (NPY), TSH and biologically active analogs thereof. The term xe2x80x9cbiologically active analogsxe2x80x9d is used herein to cover naturally occurring, recombinant, and synthetic peptides, polypeptides, and proteins having physiological or therapeutic activity. In general, the term covers all fragments and derivatives of a peptide, protein, or a polypeptide that exhibit a qualitatively similar agonist or antagonist effect to that of the unmodified, or naturally occurring peptide, protein, or polypeptide, e.g., those in which one or more of the amino acid residues occurring in the natural compounds are substituted or deleted, or in which the N- or C-terminal residues has been structurally modified. The term effective ionogenic amine refers to a free amine present on the polypeptide which is capable of forming an ionic bond with the free carboxylic groups on the copolymer.
Examples of other somatostatin analogs include, but are not limited to, the following somatostatin analogs which are disclosed in the above-cited references: H-xcex2-D-Nal-Cys-Tyr-D-Trp-Lys-Val-Cys-Thr-NH2 acetate salt (also known as SOMATULINE(trademark)), where the two Cysteines are bonded by a disulfide bond;
H-D-Phe-Cys-Phe-D-Trp-Lys-Thr-Cys-xcex2-Nal-NH2;
H-D-Phe-Cys-Tyr-D-Trp-Lys-Thr-Cys-xcex2-Nal-NH2;
H-D-xcex2-Nal-Cys-Phe-D-Trp-Lys-Thr-Cys-Thr-N H2;
H-D-Phe-Cys-Tyr-D-Trp-Lys-Thr-Pen-Thr-NH2;
H-D-Phe-Cys-Phe-D-Trp-Lys-Thr-Pen-Thr-NH2;
H-D-Phe-Cys-Tyr-D-Trp-Lys-Thr-Pen-Thr-OH;
H-D-Phe-Cys-Phe-D-Trp-Lys-Thr-Pen-Thr-OH;
H-Gly-Pen-Phe-D-Trp-Lys-Thr-Cys-Thr-OH;
H-Phe-Pen-Tyr-D-Trp-Lys-Thr-Cys-Thr-OH;
H-Phe-Pen-Phe-D-Trp-Lys-Thr-Pen-Thr-OH;
H-D-Phe-Cys-Phe-D-Trp-Lys-Thr-Cys-Thr-ol;
H-D-Phe-Cys-Phe-D-Trp-Lys-Thr-Cys-Thr-NH2;
H-D-Trp-Cys-Tyr-D-Trp-Lys-Val-Cys-Thr-NH2;
H-D-Trp-Cys-Phe-D-Trp-Lys-Thr-Cys-Thr-N H2;
H-D-Phe-Cys-Tyr-D-Trp-Lys-Val-Cys-Thr-NH2;
H-D-Phe-Cys-Tyr-D-Trp-Lys-Val-Cys-Trp-N H2;
H-D-Phe-Cys-Tyr-D-Trp-Lys-Val-Cys-Thr-NH2;
Ac-D-Phe-Lys*-Tyr-D-Trp-Lys-Val-Asp*-Thr-NH2 (an amide bridge formed between Lys* and Asp*);
Ac-hArg(Et)2-Gly-Cys-Phe-D-Trp-Lys-Thr-Cys-Thr-NH2;
Ac-D-hArg(Et)2-Gly-Cys-Phe-D-Trp-Lys-Thr-Cys-Thr-NH2;
Ac-D-hArg(Bu)-Gly-Cys-Phe-D-Trp-Lys-Thr-Cys-Thr-NH2;
Ac-D-hArg(Et)2-Cys-Phe-D-Trp-Lys-Thr-Cys-Thr-NH2;
Ac-L-hArg(Et)2-Cys-Phe-D-Trp-Lys-Thr-Cys-Thr-N H2;
Ac-D-hArg(CH2CF3)2-CYS-Phe-D-Trp-Lys-Thr-Cys-Thr-NH2;
Ac-D-hArg(CH2CF3)2-Gly-Cys-Phe-D-Trp-Lys-Thr-Cys-Thr-NH2;
Ac-D-hArg(CH2CF3)2-Gly-Cys-Phe-D-Trp-Lys-Thr-Cys-Phe-NH2;
Ac-D-hArg(CH2CF3)2-Gly-Cys-Phe-D-Trp-Lys-Thr-Cys-Thr-NHEt;
Ac-L-hArg(CH2-CF3)2-Gly-Cys-Phe-D-Trp-Lys-Thr-Cys-Thr-NH2;
Ac-D-hArg(CH2CF3)2-Gly-Cys-Phe-D-Trp-Lys(Me)-Thr-Cys-Thr-NH2;
Ac-D-hArg(CH2CF3)2-Gly-Cys-Phe-D-Trp-Lys(Me)-Thr-Cys-Thr-NHEt;
Ac-hArg(CH3, hexyl)-Gly-Cys-Phe-D-Trp-Lys-Thr-Cys-Thr-NH2;
H-hArg(hexyl2)-Gly-Cys-Phe-D-Trp-Lys-Thr-Cys-Thr-NH2;
Ac-D-hArg(Et)2-Gly-Cys-Phe-D-Trp-Lys-Thr-Cys-Thr-NHEt;
Ac-D-hArg(Et)2-Gly-Cys-Phe-D-Trp-Lys-Thr-Cys-Phe-NH2;
Propionyl-D-hArg(Et)2-Gly-Cys-Phe-D-Trp-Lys(iPr)-Thr-Cys-Thr-NH2;
Ac-D-xcex2-Nal-Gly-Cys-Phe-D-Trp-Lys-Thr-Cys-Gly-hArg(Et)2-NH2;
Ac-D-Lys(iPr)-Gly-Cys-Phe-D-Trp-Lys-Thr-Cys-Thr-NH2;
Ac-D-hArg(CH2CF3)2-D-hArg(CH2CF3)2-Gly-Cys-Phe-D-Trp-Lys-Thr-Cys-Thr-NH2;
Ac-D-hArg(CH2CF3)2-D-hArg(CH2CF3)2-Gly-Cys-Phe-D-Trp-Lys-Thr-Cys-Phe-NH2;
Ac-D-hArg(Et)2-D-hArg(Et)2-Gly-Cys-Phe-D-Trp-Lys-Thr-Cys-Thr-NH2;
Ac-Cys-Lys-Asn-4-Cl-Phe-Phe-D-Trp-Lys-Thr-Phe-Thr-Ser-D-Cys-NH2;
H-Bmp-Tyr-D-Trp-Lys-Val-Cys-Thr-NH2;
H-Bmp-Tyr-D-Trp-Lys-Val-Cys-Phe-NH2;
H-Bmp-Tyr-D-Trp-Lys-Val-Cys-p-C-Phe-N H2;
H-Bmp-Tyr-D-Trp-Lys-Val-Cys-xcex2-Nal-NH2;
H-D-xcex2-Nal-Cys-Tyr-D-Trp-Lys-Val-Cys-Thr-NH2;
H-D-Phe-Cys-Tyr-D-Trp-Lys-Abu-Cys-Thr-NH2;
H-D-Phe-Cys-Tyr-D-Trp-Lys-Abu-Cys-xcex2-Nal-NH2;
H-pentafluoro-D-Phe-Cys-Tyr-D-Trp-Lys-Val-Cys-Thr-NH2;
Ac-D-xcex2-Nal-Cys-pentafluoro-Phe-D-Trp-Lys-Val-Cys-Thr-NH2;
H-D-xcex2-Nal-Cys-Tyr-D-Trp-Lys-Val-Cys-xcex2-Nal-NH2;
H-D-Phe-Cys-Tyr-D-Trp-Lys-Val-Cys-xcex2-Nal-NH2;
H-D-xcex2-Nal-Cys-Tyr-D-Trp-Lys-Abu-Cys-Thr-NH2;
H-D-p-Cl-Phe-Cys-Tyr-D-Trp-Lys-Abu-Cys-Thr-NH2;
Ac-D-p-Cl-Phe-Cys-Tyr-D-Trp-Lys-Abu-Cys-Thr-NH2;
H-D-Phe-Cys-xcex2-Nal-D-Trp-Lys-Val-Cys-Thr-NH2;
H-D-Phe-Cys-Tyr-D-Trp-Lys-Cys-Thr-NH2;
cyclo(Pro-Phe-D-Trp-N-Me-Lys-Thr-Phe);
cyclo(Pro-Phe-D-Trp-N-Me-Lys-Thr-Phe);
cyclo(Pro-Phe-D-Trp-Lys-Thr-N-Me-Phe);
cyclo(N-Me-Ala-Tyr-D-Trp-Lys-Thr-Phe);
cyclo(Pro-Tyr-D-Trp-Lys-Thr-Phe);
cyclo(Pro-Phe-D-Trp-Lys-Thr-Phe);
cyclo(Pro-Phe-L-Trp-Lys-Thr-Phe);
cyclo(Pro-Phe-D-Trp(F)-Lys-Thr-Phe);
cyclo(Pro-Phe-Trp(F)-Lys-Thr-Phe);
cyclo(Pro-Phe-D-Trp-Lys-Ser-Phe);
cyclo(Pro-Phe-D-Trp-Lys-Thr-p-Cl-Phe);
cyclo(D-Ala-N-Me-D-Phe-D-Thr-D-Lys-Trp-D-Phe);
cyclo(D-Ala-N-Me-D-Phe-D-Val-Lys-D-Trp-D-Phe);
cyclo(D-Ala-N-Me-D-Phe-D-Thr-Lys-D-Trp-D-Phe);
cyclo(D-Abu-N-Me-D-Phe-D-Val-Lys-D-Trp-D-Tyr);
cyclo(Pro-Tyr-D-Trp-t-4-AchxAla-Thr-Phe);
cyclo(Pro-Phe-D-Trp-t-4-AchxAla-Thr-Phe);
cyclo(N-Me-Ala-Tyr-D-Trp-Lys-Val-Phe);
cyclo(N-Me-Ala-Tyr-D-Trp-t-4-AchxAla-Thr-Phe);
cyclo(Pro-Tyr-D-Trp-4-Amphe-Thr-Phe);
cyclo(Pro-Phe-D-Trp-4-Amphe-Thr-Phe);
cyclo(N-Me-Ala-Tyr-D-Trp-4-Amphe-Thr-Phe);
cyclo(Asn-Phe-Phe-D-Trp-Lys-Thr-Phe-Gaba);
cyclo(Asn-Phe-Phe-D-Trp-Lys-Thr-Phe-Gaba-Gaba);
cyclo(Asn-Phe-D-Trp-Lys-Thr-Phe);
cyclo(Asn-Phe-Phe-D-Trp-Lys-Thr-Phe-NH(CH2)4CO);
cyclo(Asn-Phe-Phe-D-Trp-Lys-Thr-Phe-xcex2-Ala);
cyclo(Asn-Phe-Phe-D-Trp-Lys-Thr-Phe-D-Glu)-OH;
cyclo(Phe-Phe-D-Trp-Lys-Thr-Phe);
cyclo(Phe-Phe-D-Trp-Lys-Thr-Phe-Gly)
cyclo(Phe-Phe-D-Trp-Lys-Thr-Phe-Gaba);
cyclo(Asn-Phe-Phe-D-Trp-Lys-Thr-Phe-Gly);
cyclo(Asn-Phe-Phe-D-Trp(F)-Lys-Thr-Phe-Gaba);
cyclo(Asn-Phe-Phe-D-Trp(NO2)-Lys-Thr-Phe-Gaba);
cyclo(Asn-Phe-Phe-Trp(Br)-Lys-Thr-Phe-Gaba);
cyclo(Asn-Phe-Phe-D-Trp-Lys-Thr-Phe(I)-Gaba);
cyclo(Asn-Phe-Phe-D-Trp-Lys-Thr-Tyr(But)-Gaba);
cyclo(Bmp-Lys-Asn-Phe-Phe-D-Trp-Lys-Thr-Phe-Thr-Pro-Cys)xe2x80x94OH;
cyclo(Bmp-Lys-Asn-Phe-Phe-D-Trp-Lys-Thr-Phe -Thr-Pro-Cys)xe2x80x94OH;
cyclo(Bmp-Lys-Asn-Phe-Phe-D-Trp-Lys -Thr-Phe-Thr-Tpo-Cys)xe2x80x94OH;
cyclo(Bmp-Lys-Asn-Phe-Phe-D-Trp-Lys-Thr-Phe-Thr-MeLeu-Cys)xe2x80x94OH;
cyclo(Phe-Phe-D-Trp-Lys-Thr-Phe-Phe-Gaba);
cyclo(Phe-Phe-D-Trp-Lys-Thr-Phe-D-Phe-Gaba);
cyclo(Phe-Phe-D-Trp(5F)-Lys-Thr-Phe-Phe-Gaba);
cyclo(Asn-Phe-Phe-D-Trp-Lys(Ac)-Thr-Phe-NH-(CH2)3xe2x80x94CO);
cyclo(Lys-Phe-Phe-D-Trp-Lys-Thr-Phe-Gaba);
cyclo(Lys-Phe-Phe-D-Trp-Lys-Thr-Phe-Gaba);
cyclo(Orn-Phe-Phe-D-Trp-Lys-Thr-Phe-Gaba);
H-Cys-Phe-Phe-D-Trp-Lys-Thr-Phe-Cys-NH2;
H-Cys-Phe-Phe-D-Trp-Lys-Ser-Phe-Cys-NH2;
H-Cys-Phe-Tyr-D-Trp-Lys-Thr-Phe-Cys-NH2; and
H-Cys-Phe-Tyr(I)-D-Trp-Lys-Thr-Phe-Cys-NH2.
A disulfide bridge is formed between the two free thiols (e.g., Cys, Pen, or Bmp residues) when they are present in a peptide; however, the disulfide bond is not shown.
Also included are somatostatin agonists of the following formula: 
wherein
A1 is a D- or L-isomer of Ala, Leu, lie, Val, Nle, Thr, Ser, xcex2-Nal, xcex2-Pal, Trp, Phe, 2,4-dichloro-Phe, pentafluoro-Phe, p-X-Phe, or o-X-Phe, wherein X is CH3, Cl, Br, F, OH, OCH3 or NO2;
A2 is Ala, Leu, lie, Val, Nle, Phe, xcex2-Nal, pyridyl-Ala, Trp, 2,4-dichloro-Phe, pentafluoro-Phe, o-X-Phe, or p-X-Phe, wherein X is CH3, Cl, Br, F, OH, OCH3 or NO2;
A3 is pyridyl-Ala, Trp, Phe, xcex2-Nal, 2,4-dichloro-Phe, pentafluoro-Phe, o-X-Phe, or p-X-Phe, wherein X is CH3, Cl, Br, F, OH, OCH3 or NO2;
A6 is Val, Ala, Leu, lie, Nle, Thr, Abu, or Ser;
A7 is Ala, Leu, lie, Val, Nle, Phe, xcex2-Nal, pyridyl-Ala, Trp, 2,4-dichloro-Phe, pentafluoro-Phe, o-X-Phe, or p-X-Phe, wherein X is CH3, Cl, Br, F, OH, OCH3 or NO2;
A8 is a D- or L-isomer of Ala, Leu, lie, Val, Nle, Thr, Ser, Phe, xcex2-Nal, pyridyl-Ala, Trp, 2,4-dichloro-Phe, pentafluoro-Phe, p-X-Phe, or o-X-Phe, wherein X is CH3, Cl, Br, F, OH, OCH3 or NO2;
each R1 and R2, independently, is H, lower acyl or lower alkyl; and R3 is OH or NH2; provided that at least one of A1 and A8 and one of A2 and A7 must be an aromatic amino acid; and further provided that A1, A2, A7 and A8 cannot all be aromatic amino acids.
Examples of linear agonists to be used in a process of this invention include:
H-D-Phe-p-chloro-Phe-Tyr-D-Trp-Lys-Thr-Phe-Thr-NH2;
H-D-Phe-p-NO2-Phe-Tyr-D-Trp-Lys-Val-Phe-Thr-NH2;
H-D-Nal-p-chloro-Phe-Tyr-D-Trp-Lys-Val-Phe-Thr-N H2;
H-D-Phe-Phe-Phe-D-Trp-Lys-Thr-Phe-Thr-NH2;
H-D-Phe-Phe-Tyr-D-Trp-Lys-Val-Phe-Thr-NH2;
H-D-Phe-p-chloro-Phe-Tyr-D-Trp-Lys-Val-Phe-Thr-NH2; and
H-D-Phe-Ala-Tyr-D-Trp-Lys-Val-Ala-xcex2-D-Nal-NH2.
If desired, one or more chemical moieties, e.g., a sugar derivative, mono or poly-hydroxy C2-12 alkyl, mono or poly-hydroxy C2-12 acyl groups, or a piperazine derivative, can be attached to the somatostatin agonist, e.g., to the N-terminus amino acid. See PCT Application WO 88/02756, European Application 0 329 295, and PCT Application No. WO 94/04752. An example of somatostatin agonists which contain N-terminal chemical substitutions are: 
or a pharmaceutically acceptable salt thereof.
Examples of specific LHRH analogues that can be incorporated in a conjugate or composition of this invention are TRYPTORELIN(trademark) (p-Glu-His-Trp-Ser-Tyr-D-Trp-Leu-Arg-Pro-Gly-NH2), buserelin ([D-Ser(t-Bu)6, des-Gly-NH210]-LHRH(1-9)NHEt), deslorelin ([D-Trp6, des-Gly-NH210]-LHRH(1-9)NHEt, fertirelin ([des-Gly-NH210]-LHRH(1-9)NHEt), gosrelin ([D-Ser(t-Bu)6, Azgly10]-LHRH), histrelin ([D-His(Bzl)6, des-Gly-NH210]-LHRH(1-9)NHEt), leuprorelin ([D-Leu6, des-Gly-NH210]-LHRH(1-9)NHEt), lutrelin ([D-Trp6, MeLeu7, des-Gly-NH210]-LHRH(1-9)NHEt), nafarelin ([D-Nal6]-LHRH and pharmaceutically acceptable salts thereof.
The release of the polypeptide from the composition may be modified by changing the chemical structure of the composition. Increasing the molecular weight of the polymer will decrease the rate of peptide released from the conjugate. Increasing the number of carboxylic acid groups on the polymer will increase the amount of polypeptide ionically bound to the composition, and consequently, increase the amount of release of the peptide from the conjugate.
The release of the polypeptide may be further modulated through (a) treating the composition with soluble salts of divalent or polyvalent metallic ions of weak acids (e.g., calcium, iron, magnesium, or zinc); (b) coating the particles with a thin, absorbable layer made of a glycolide copolymer or silicone oil in a spherical, cylindrical, or planar configuration; or (c) microencapsulating the composition in an absorbable glycolide copolymer. In one embodiment, the composition comprises between 0.01 and 20 percent, by weight, of a polyvalent metal.
Depending on the choice of polypeptide, the compositions can be used to treat any number of disorders. For example, somatostatin, bombesin, GRP, LHRH, and analogs thereof, have been shown to treat various forms of cancer. Growth factors such as GH, GRF, and GHRP, and analogs thereof, have been shown to stimulate growth in both adolescents and the elderly. Calcitonin, amylin, PTH, and PTHrP, and analogs thereof, have been shown to treat osteoporosis and other bone disorders.
The compositions are designed for parenteral administration, e.g., intramuscular, subcutaneous, intradural, or intraperitoneal injection. Preferably, the compositions are administered intramuscularly.
The compositions of the invention can be in the form of powder or a microparticle to be administered as a suspension with a pharmaceutically acceptable vehicle (e.g., water with or without a carrier substance such as mannitol or polysorbate). The compositions may also be compounded in the form of a rod for parenteral implantation using a trocar, e.g., intramuscular implantation.
The dose of the composition of the present invention for treating the abovementioned diseases or disorders varies depending upon the manner of administration, the age and the body weight of the subject, and the condition of the subject to be treated, and ultimately will be decided by the attending physician or veterinarian. Such an amount of the composition as determined by the attending physician or veterinarian is referred to herein as a xe2x80x9ctherapeutically effective amount.xe2x80x9d
In another aspect, the present invention features a process of synthesizing a copolymer, the process comprising the steps of: reacting chitosan with a weak acid to produce a lower molecular weight polysaccharide; reacting between 1 and 50 percent of the free amines of the lower molecular weight polysaccharide with a first acylating agent, the first acylating agent selected from the group consisting of C4-C34 polycarboxyalkane, C4-C34 polycarboxyalkene, C8-C40 polycarboxyarylalkane, C10-C40 polycarboxyarylalkene, or an acylating derivative thereof; and reacting between 50 and 100 percent of the free amine of the lower molecular weight polysaccharide with a second acylating agent, the second acylating agent selected from the group consisting of C2-31 monocarboxyalkane, C3-31 monocarboxyalkene, C7-38 monocarboxyarylalkane, C9-35 monocarboxyarylalkene, or an acylating derivative thereof. The reaction of the lower molecular weight polysaccharide with both the first acylating agent and the second acylating agent may be measured with an amine detecting agent (e.g., fluorescamine) to ensure that between 1 and 50 percent of the free amines of the lower molecular weight polysaccharide are acylated with the first acylating agent and between 50 and 99 percent of the free amines of the lower molecular weight polysaccharide are acylated with the second acylating agent. See. e.g., Bailey, P. D., An Introduction to Peptide Chemistry (Wiley, N.Y.)(1990); Oppenheimer, H, et al. Archives Biochem. Biophys. 120:108-118 (1967); Stein, S, Arch. Biochem. Biophys. 155:203-212 (1973).
Reacting chitosan with the weak acid (e.g., nitrous acid) cleaves the polymer, thereby reducing its molecular weight (e.g., 2,500-80,000 daltons). In preferred embodiments, the first acylating group and the second acylating agent are reacted with the lower molecular weight polysaccharide successively, e.g., either the first acylating agent is reacted before the second acylating agent is reacted or the second acylating agent is reacted before the first acylating agent or simultaneously. As a result of the acylation of the free amines, some of the free hydroxy groups of the lower molecular weight polysaccharide may be acylated. The extent of the acylation of the free hydroxy groups may be altered by changing the pH or the solvents or agents used during the acylation reactions, or the acylating agents used.
Examples of acylating derivatives include, but are not limited to, anhydrides and N-acylated heterocycles (e.g., imidazoles and pyrazoles). See e.g., Bodansky, et al., The Practice of Peptide Synthesis, 87-150 (Springer-Verlag, 1984). The agents polycarboxyalkane, polycarboxyalkene, polycarboxyarylalkane, and polycarboxyarylalkene or acylating derivatives thereof contain, or originate from reactants containing, 2-5 carboxylic acid functionalities. The substituents monocarboxyalkane, monocarboxyalkene, monocarboxyarylalkane, and monocarboxyarylalkene contain, or originate from reactants containing, only a single carboxylic acid group. Examples of first acylating agents include, but are not limited to, succinic anhydride, 2-(C1-30 alkyl)succinic anhydride, 2-(C2-30 alkenyl)succinic anhydride, maleic anhydride, glutaric anhydride, itaconic anhydride, and phthalic anhydride. Examples of second acylating agents include, but are not limited to, acetic anhydride, benzoic anhydride, N,Nxe2x80x2-diacetyl-3,5-dimethylpyrazole, N,Nxe2x80x2-diacetylimidazole, phenylacetic anhydride, propionic anhydride, and butyric anhydride.
In yet another aspect, the present invention features a process of synthesizing a composition, the process comprising the steps of: reacting chitosan with a weak acid to produce a lower molecular weight polysaccharide; reacting between 1 and 50 percent of the free amines of the lower molecular weight polysaccharide with a first acylating agent, the first acylating agent selected from the group consisting of C4-C34 polycarboxyalkane, C4-C34 polycarboxyalkene, C8-C40 polycarboxyarylalkane, C10-C40 polycarboxyarylalkene, or an acylating derivative thereof; reacting between 50 and 100 percent of the free amine of the lower molecular weight polysaccharide with a second acylating agent, the second acylating agent selected from the group consisting of C2-31 monocarboxyalkane, C3-31 monocarboxyalkene, C7-38 monocarboxyarylalkane, C9-35 monocarboxyarylalkene, or an acylating derivative thereof; neutralizing the acylated lower molecular weight polysaccharide with a base; and mixing the neutralized lower acylated molecular weight polysaccharide with a polypeptide salt, wherein the polypeptide salt comprises at least one ionogenic amine, to form a polypeptide-copolymer ionic conjugate.
The neutralization step preferably renders the lower molecular weight polysaccharide emulsifiable or soluble in water. In preferred embodiments, the base is an inorganic base (e.g., sodium hydroxide). The polypeptide salt is preferably a weak acid salt (e.g., acetate, lactate, or citrate). The ionic conjugate can be isolated by filtering or by centrifuging the resulting mixture.
The conjugates of the invention can easily be made into injectable microspheres or microparticles, and implantable films or rods, without the need to utilize processing that entails multiphase emulsions. Preferably, the microparticles are manufactured by (a) dissolving the composition in an aprotic, water miscible organic solvent; (b) mixing the organic solvent in water; and (c) isolating the microparticles from the water. In preferred embodiments, the organic solvent is chosen from the group of acetone, acetonitrile, tetrahydrofuran, dimethylformamide, and dimethyl ethylene glycol.
Other features and advantages of the present invention will be apparent from the detailed description and from the claims.